Differential refractometer for gradient chromatography

ABSTRACT

The present disclosure describes a differential refractometer for gradient chromatography. In an exemplary embodiment, the differential refractometer includes a solvent delay volume, an eluent flow meter coupled to an eluent inlet of a sample cell, a solvent flow regulator coupled to an outlet of the solvent delay volume and coupled to a solvent inlet of a reference cell, an instrument controller configured to receive the eluent flow rate from the eluent flow meter, configured to receive the solvent flow rate from the solvent flow regulator, configured to receive a flow rate ratio from a flow rate ratio data source, wherein the flow rate ratio indicates a ratio of the eluent flow rate to the solvent flow rate, and an optical bench configured to measure a difference between a refractive index of the eluent present in the sample cell and a refractive index of the solvent present in the reference cell.

PRIORITY

This application is a continuation-in-part of U.S. patent applicationSer. No. 17/383,426, filed Jul. 22, 2021.

BACKGROUND

The present disclosure relates to differential refractometry, and morespecifically, to a differential refractometer for gradientchromatography.

SUMMARY

The present disclosure describes a differential refractometer forgradient chromatography. In an exemplary embodiment, the differentialrefractometer includes (1) a solvent delay volume configured to becoupled to an outlet of a chromatography pump, (2) an eluent flow meterconfigured to be coupled to an outlet of a chromatography column andcoupled to an eluent inlet of a sample cell, where the eluent flow meteris configured to measure an eluent flow rate of an eluent flowingthrough the sample cell, (3) a solvent flow regulator coupled to anoutlet of the solvent delay volume and coupled to a solvent inlet of areference cell, where the solvent flow regulator is configured tomeasure and to regulate a solvent flow rate of a solvent flowing throughthe reference cell, (4) an instrument controller configured to receivethe eluent flow rate from the eluent flow meter, configured to receivethe solvent flow rate from the solvent flow regulator, configured toreceive a flow rate ratio from a flow rate ratio data source, whereinthe flow rate ratio indicates a ratio of the eluent flow rate to thesolvent flow rate, and configured to transmit a flow command to thesolvent flow regulator to achieve the flow rate ratio, and (5) anoptical bench configured to measure, in response to receiving from theinstrument controller a signal indicating that the flow rate ratio hasbeen achieved, a difference between a refractive index of the eluentpresent in the sample cell and a refractive index of the solvent presentin the reference cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts an existing differential refractometer.

FIG. 1B depicts an existing sample cell of a differential refractometer.

FIG. 1C depicts a graph in accordance with an existing differentialrefractometer.

FIG. 1D depicts a graph in accordance with an existing differentialrefractometer.

FIG. 2 depicts a differential refractometer in accordance with anexemplary embodiment.

FIG. 3A depicts a differential refractometer in accordance with anembodiment.

FIG. 3B depicts a sample cell of a differential refractometer inaccordance with an embodiment.

FIG. 4A depicts a graph in accordance with an embodiment.

FIG. 4B depicts a graph in accordance with an embodiment.

FIG. 5 depicts a computer system in accordance with an exemplaryembodiment.

DETAILED DESCRIPTION

The present disclosure describes a differential refractometer forgradient chromatography. In an exemplary embodiment, the differentialrefractometer includes (1) a solvent delay volume configured to becoupled to an outlet of a chromatography pump, (2) an eluent flow meterconfigured to be coupled to an outlet of a chromatography column andcoupled to an eluent inlet of a sample cell, where the eluent flow meteris configured to measure an eluent flow rate of an eluent flowingthrough the sample cell, (3) a solvent flow regulator coupled to anoutlet of the solvent delay volume and coupled to a solvent inlet of areference cell, where the solvent flow regulator is configured tomeasure and to regulate a solvent flow rate of a solvent flowing throughthe reference cell, (4) an instrument controller configured to receivethe eluent flow rate from the eluent flow meter, configured to receivethe solvent flow rate from the solvent flow regulator, configured toreceive a flow rate ratio from a flow rate ratio data source, whereinthe flow rate ratio indicates a ratio of the eluent flow rate to thesolvent flow rate, and configured to transmit a flow command to thesolvent flow regulator to achieve the flow rate ratio, and (5) anoptical bench configured to measure, in response to receiving from theinstrument controller a signal indicating that the flow rate ratio hasbeen achieved, a difference between a refractive index of the eluentpresent in the sample cell and a refractive index of the solvent presentin the reference cell. In an embodiment, the flow rate ratio is a ratioof a volume of a void in the chromatography column to a volume of a voidin the solvent delay column. In an embodiment, the flow rate ratio is aratio of a sum of a volume of a void in the chromatography column, avolume of tubing coupled to the chromatography column, and a volume ofthe eluent flow meter, to a sum of a volume of a void in the solventdelay column, a volume of tubing coupled to the solvent delay column,and a volume of the solvent flow regulator.

In an exemplary embodiment, the differential refractometer includes (1)a solvent delay volume configured to be coupled to an outlet of achromatography pump, (2) an eluent flow meter configured to be coupledto an outlet of a chromatography column and coupled to an eluent inletof a sample cell, where the eluent flow meter is configured to measurean eluent flow rate of an eluent flowing through the sample cell, (3) asolvent flow regulator coupled to an outlet of the solvent delay volumeand coupled to a solvent inlet of a reference cell, where the solventflow regulator is configured to measure and to regulate a solvent flowrate of a solvent flowing through the reference cell, and (4) aninstrument controller configured to receive the eluent flow rate fromthe eluent flow meter, configured to receive the solvent flow rate fromthe solvent flow regulator, configured to receive a flow rate ratio froma flow rate ratio data source, wherein the flow rate ratio indicates aratio of the eluent flow rate to the solvent flow rate, and configuredto transmit a flow command to the solvent flow regulator to achieve theflow rate ratio. In a further embodiment, the differential refractometerfurther includes an optical bench configured to measure, in response toreceiving from the instrument controller a signal indicating that theflow rate ratio has been achieved, a difference between a refractiveindex of the eluent present in the sample cell and a refractive index ofthe solvent present in the reference cell. In an embodiment, the flowrate ratio is a ratio of a volume of a void in the chromatography columnto a volume of a void in the solvent delay column. In an embodiment, theflow rate ratio is a ratio of a sum of a volume of a void in thechromatography column, a volume of tubing coupled to the chromatographycolumn, and a volume of the eluent flow meter, to a sum of a volume of avoid in the solvent delay column, a volume of tubing coupled to thesolvent delay column, and a volume of the solvent flow regulator.

Definitions

Particle

A particle may be a constituent of a liquid sample aliquot. Suchparticles may be molecules of varying types and sizes, nanoparticles,virus like particles, liposomes, emulsions, bacteria, and colloids.These particles may range in size on the order of nanometer to microns.

Analysis of Macromolecular or Particle Species in Solution

The analysis of macromolecular or particle species in solution may beachieved by preparing a sample in an appropriate solvent and theninjecting an aliquot thereof into a separation system such as a liquidchromatography (LC) column or field flow fractionation (FFF) channelwhere the different species of particles contained within the sample areseparated into their various constituencies. Once separated, generallybased on size, mass, or column affinity, the samples may be subjected toanalysis by means of light scattering, refractive index, ultravioletabsorption, electrophoretic mobility, and viscometric response.

Concentration Detector

Differential Refractive Index Detector

A differential refractive index detector (dRI), or differentialrefractometer, or refractive index detector (RI or RID), is a detectorthat measures the refractive index of an analyte relative to thesolvent. They are often used as detectors for high-performance liquidchromatography and size exclusion chromatography. dRIs are considered tobe universal detectors because they can detect anything with arefractive index different from the solvent, but they have lowsensitivity. When light leaves one material and enters another it bends,or refracts. The refractive index of a material is a measure of how muchlight bends when it enters.

A differential refractive index detector contain a flow cell with thefollowing two parts: one for the sample; and one for the referencesolvent. The dRI measures the refractive index of both components. Whenonly solvent is passing through the sample component, the measuredrefractive index of both components is the same, but when an analytepasses through the flow cell, the two measured refractive indices aredifferent. The difference appears as a peak in the chromatogram.Differential refractive index detectors are often used for the analysisof polymer samples in size exclusion chromatography. A dRI could outputa concentration detector signal value corresponding to a concentrationvalue of a sample.

dRI instruments, which measure small changes in refractive index of asolution, are highly desirable for measuring analyte concentration insolutions (e.g., in a chromatographic or FFF separation). dRIinstruments are commonly used for isocratic chromatography, where thesolvent is constant, and therefore has a constant refractive index,throughout the separation. Since dRI measurement does not require theanalyte to contain chromophores or fluorophores, dRI measurement issuitable for a very wide range of molecular or macromolecular analytesincluding dissolved salts, polysaccharides, synthetic polymers,proteins, nucleic acids, lipids and more. dRI measurement offers a verywide range of concentration measurements, from ng/mL to g/mL.

dRI measurement involves measuring the difference in refractive indexbetween (a) the solution containing solvent and analyte, located in thesample cell, and (b) the solvent alone located in the reference cell.The solution in the sample cell is usually changing (e.g., as eluentflows from a chromatographic separation system or another source ofsample solution, the quantity of dissolved analyte changes). The solventin the reference cell is not changed in the course of the measurement.Hence if, in the sample cell, the solvent is constant and only theanalyte concentration changes, as in an isocratic separation, dRIprovides a signal proportional to the analyte concentration.

Gradient Chromatography

Gradient chromatography involves mixing two solutions to provide aseries of mobile phases that enhance macromolecular separation.Typically the different mixtures have different refractive indices,leading to large changes in dRI signals, which leads to error in analyteconcentration measurement or even obscuration of the analyte dRI signal.Commonly, macromolecules such as proteins or polysaccharides areseparated by gradient chromatography.

Volumes in Chromatography Columns

The void volume or total exclusion volume is the interstitial volumebetween the packed beads in a chromatography column. The total inclusionvolume is the combined value or sum of the volume of all the pores inthe packed beads in a chromatography column and the interstitial volumeof the chromatography column.

Current Technologies

Current technologies, as depicted in FIG. 1A, FIG. 1B, FIG. 1C, and FIG.1D, depict how, if the solvent in the sample cell is also changing(e.g., as in a gradient chromatography such as ion-exchangechromatography (changing salt concentration) or reverse-phasechromatography (changing ratio between polar and non-polar solvent)),dRI provides a signal proportional to both the analyte concentration andchanging solvent composition, which is not useful for determininganalyte concentration. Thus, there is a need a differentialrefractometer for gradient chromatography.

Referring to FIG. 2 , in an exemplary embodiment, the differentialrefractometer includes (1) a solvent delay volume 210 configured to becoupled to an outlet of a chromatography pump 202, (2) an eluent flowmeter 220 configured to be coupled to an outlet of a chromatographycolumn 204 and coupled to an eluent inlet of a sample cell 230, whereeluent flow meter 220 is configured to measure an eluent flow rate of aneluent flowing through sample cell 230, (3) a solvent flow regulator 240coupled to an outlet of solvent delay volume 210 and coupled to asolvent inlet of a reference cell 250, where solvent flow regulator 240is configured to measure and to regulate a solvent flow rate of asolvent flowing through reference cell 250, (4) an instrument controller260 configured to receive the eluent flow rate from eluent flow meter220, configured to receive the solvent flow rate from solvent flowregulator 240, configured to receive a flow rate ratio from a flow rateratio data source 206, wherein the flow rate ratio indicates a ratio ofthe eluent flow rate to the solvent flow rate, and configured totransmit a flow command to solvent flow regulator 240 to achieve theflow rate ratio, and (5) an optical bench 270 configured to measure, inresponse to receiving from instrument controller 260 a signal indicatingthat the flow rate ratio has been achieved, a difference between arefractive index of the eluent present in sample cell 230 and arefractive index of the solvent present in reference cell 250.

In an embodiment, the flow rate ratio is a ratio of a volume of a voidin chromatography column 204 to a volume of a void in solvent delaycolumn 210. In an embodiment, the flow rate ratio is a ratio of a sum ofa volume of a void in chromatography column 204, a volume of tubingcoupled to chromatography column 204, and a volume of eluent flow meter220, to a sum of a volume of a void in solvent delay column 210, avolume of tubing coupled to solvent delay column 210, and a volume ofsolvent flow regulator 240.

FIG. 3A depicts a differential refractometer in accordance with anembodiment. FIG. 3B depicts a sample cell of a differentialrefractometer in accordance with an embodiment.

In an exemplary embodiment, instrument controller 260 is a standalonecomputer system, such as computer system 500 shown in FIG. 5 , a networkof distributed computers, where at least some of the computers arecomputer systems such as computer system 500 shown in FIG. 5 , or acloud computing node server, such as computer system 500 shown in FIG. 5. In an embodiment, instrument controller 260 is a computer system 500as shown in FIG. 5 , that executes a differential refractometer forgradient chromatography script or computer software application thatcarries out the operations of at least a method carried out byinstrument controller 260. In an embodiment, instrument controller 260is a computer system/server 512 as shown in FIG. 5 , that executes adifferential refractometer for gradient chromatography script orcomputer software application that carries out the operations of atleast a method carried out by instrument controller 260. In anembodiment, instrument controller 260 is a processing unit 516 as shownin FIG. 5 , that executes a differential refractometer for gradientchromatography script or computer software application that carries outthe operations of at least a method carried out by instrument controller260.

For example, reference cell 250 is provided with a changing solvent,matched to the changing solvent in sample cell 230 by splitting the flowemerging from the solvent mixing device, mixer 203, prior to itsentering the sample loop, sending one portion to reference cell 250 andthe remainder to continue within the chromatography system passingthrough the sample loop and separation column, chromatography column204, before entering the dRI instrument's sample cell 230. In a furtherexample, the portion of solvent destined for the dRI instrument'sreference cell 250 passes through a delay volume, solvent delay volume210, thereby inducing a timing delay equivalent to the delayexperienced, upon passage through the chromatographic column,chromatography column 204, by the portion of solvent destined for samplecell 230. In this manner, for example, at each point in time, the samesolvent composition would be present in sample cell 230 and referencecell 250, and the differential RI would be proportional only to theanalyte concentration. In addition, for example, in order to maintaincorrect and constant flow rates through the chromatographic column,chromatography column 204, and the delay column, solvent delay column210, a flow meter, eluent flow meter 220, is placed in-line with thechromatography flow path, and a flow regulator, solvent flow regulator240, is placed in-line with the solvent delay flow path. For example, ifsolvent delay column 210 were to contain one tenth of the volume ofchromatography column 204, the flow rate through solvent delay column210 is maintained at one tenth of the flow rate though chromatographycolumn 204.

In an embodiment, the flow rate ratio is a ratio of a void volume in thechromatography column to a void volume in the solvent delay column. Inan embodiment, the flow rate ratio is a ratio of a sum of a void volumein the chromatography column, a volume of tubing coupled to thechromatography column, and a volume of the eluent flow meter, to a sumof a void volume in the solvent delay column, a volume of tubing coupledto the solvent delay column, and a volume of the solvent flow regulator.

In an embodiment, the flow rate ratio is a ratio of a total inclusionvolume in the chromatography column to a total inclusion volume in thesolvent delay column. In an embodiment, the flow rate ratio is a ratioof a sum of a total inclusion volume in the chromatography column, avolume of tubing coupled to the chromatography column, and a volume ofthe eluent flow meter, to a sum of a total inclusion volume in thesolvent delay column, a volume of tubing coupled to the solvent delaycolumn, and a volume of the solvent flow regulator.

Example

For example, FIG. 4A and FIG. 4B depict how well the discloseddifferential refractometer determines analyte concentration.

Computer System

In an exemplary embodiment, the computer system is a computer system 500as shown in FIG. 5 . Computer system 500 is only one example of acomputer system and is not intended to suggest any limitation as to thescope of use or functionality of embodiments of the present invention.Regardless, computer system 500 is capable of being implemented toperform and/or performing any of the functionality/operations of thepresent invention.

Computer system 500 includes a computer system/server 512, which isoperational with numerous other general purpose or special purposecomputing system environments or configurations. Examples of well-knowncomputing systems, environments, and/or configurations that may besuitable for use with computer system/server 512 include, but are notlimited to, personal computer systems, server computer systems, thinclients, thick clients, hand-held or laptop devices, multiprocessorsystems, microprocessor-based systems, set top boxes, programmableconsumer electronics, network PCs, minicomputer systems, mainframecomputer systems, and distributed cloud computing environments thatinclude any of the above systems or devices.

Computer system/server 512 may be described in the general context ofcomputer system-executable instructions, such as program modules, beingexecuted by a computer system. Generally, program modules may includeroutines, programs, objects, components, logic, and/or data structuresthat perform particular tasks or implement particular abstract datatypes. Computer system/server 512 may be practiced in distributed cloudcomputing environments where tasks are performed by remote processingdevices that are linked through a communications network. In adistributed cloud computing environment, program modules may be locatedin both local and remote computer system storage media including memorystorage devices.

As shown in FIG. 5 , computer system/server 512 in computer system 500is shown in the form of a general-purpose computing device. Thecomponents of computer system/server 512 may include, but are notlimited to, one or more processors or processing units 516, a systemmemory 528, and a bus 518 that couples various system componentsincluding system memory 528 to processor 516.

Bus 518 represents one or more of any of several types of busstructures, including a memory bus or memory controller, a peripheralbus, an accelerated graphics port, and a processor or local bus usingany of a variety of bus architectures. By way of example, and notlimitation, such architectures include Industry Standard Architecture(ISA) bus, Micro Channel Architecture (MCA) bus, Enhanced ISA (EISA)bus, Video Electronics Standards Association (VESA) local bus, andPeripheral Component Interconnects (PCI) bus.

Computer system/server 512 typically includes a variety of computersystem readable media. Such media may be any available media that isaccessible by computer system/server 512, and includes both volatile andnon-volatile media, removable and non-removable media.

System memory 528 can include computer system readable media in the formof volatile memory, such as random access memory (RAM) 530 and/or cachememory 532. Computer system/server 512 may further include otherremovable/non-removable, volatile/non-volatile computer system storagemedia. By way of example only, storage system 534 can be provided forreading from and writing to a non-removable, non-volatile magnetic media(not shown and typically called a “hard drive”). Although not shown, amagnetic disk drive for reading from and writing to a removable,non-volatile magnetic disk (e.g., a “floppy disk”), and an optical diskdrive for reading from or writing to a removable, non-volatile opticaldisk such as a CD-ROM, DVD-ROM or other optical media can be provided.In such instances, each can be connected to bus 518 by one or more datamedia interfaces. As will be further depicted and described below,memory 528 may include at least one program product having a set (e.g.,at least one) of program modules that are configured to carry out thefunctions/operations of embodiments of the invention.

Program/utility 540, having a set (at least one) of program modules 542,may be stored in memory 528 by way of example, and not limitation.Exemplary program modules 542 may include an operating system, one ormore application programs, other program modules, and program data. Eachof the operating system, one or more application programs, other programmodules, and program data or some combination thereof, may include animplementation of a networking environment. Program modules 542generally carry out the functions and/or methodologies of embodiments ofthe present invention.

Computer system/server 512 may also communicate with one or moreexternal devices 514 such as a keyboard, a pointing device, a display524, one or more devices that enable a user to interact with computersystem/server 512, and/or any devices (e.g., network card, modem, etc.)that enable computer system/server 512 to communicate with one or moreother computing devices. Such communication can occur via Input/Output(I/O) interfaces 522. Still yet, computer system/server 512 cancommunicate with one or more networks such as a local area network(LAN), a general wide area network (WAN), and/or a public network (e.g.,the Internet) via network adapter 520. As depicted, network adapter 520communicates with the other components of computer system/server 512 viabus 518. It should be understood that although not shown, other hardwareand/or software components could be used in conjunction with computersystem/server 512. Examples, include, but are not limited to: microcode,device drivers, redundant processing units, external disk drive arrays,RAID systems, tape drives, and data archival storage systems.

Computer Program Product

The present invention may be a system, a method, and/or a computerprogram product. The computer program product may include a computerreadable storage medium (or media) having computer readable programinstructions thereon for causing a processor to carry out aspects of thepresent invention.

The computer readable storage medium can be a tangible device that canretain and store instructions for use by an instruction executiondevice. The computer readable storage medium may be, for example, but isnot limited to, an electronic storage device, a magnetic storage device,an optical storage device, an electromagnetic storage device, asemiconductor storage device, or any suitable combination of theforegoing. A non-exhaustive list of more specific examples of thecomputer readable storage medium includes the following: a portablecomputer diskette, a hard disk, a random access memory (RAM), aread-only memory (ROM), an erasable programmable read-only memory (EPROMor Flash memory), a static random access memory (SRAM), a portablecompact disc read-only memory (CD-ROM), a digital versatile disk (DVD),a memory stick, a floppy disk, a mechanically encoded device such aspunch-cards or raised structures in a groove having instructionsrecorded thereon, and any suitable combination of the foregoing. Acomputer readable storage medium, as used herein, is not to be construedas being transitory signals per se, such as radio waves or other freelypropagating electromagnetic waves, electromagnetic waves propagatingthrough a waveguide or other transmission media (e.g., light pulsespassing through a fiber-optic cable), or electrical signals transmittedthrough a wire.

Computer readable program instructions described herein can bedownloaded to respective computing/processing devices from a computerreadable storage medium or to an external computer or external storagedevice via a network, for example, the Internet, a local area network, awide area network and/or a wireless network. The network may comprisecopper transmission cables, optical transmission fibers, wirelesstransmission, routers, firewalls, switches, gateway computers and/oredge servers. A network adapter card or network interface in eachcomputing/processing device receives computer readable programinstructions from the network and forwards the computer readable programinstructions for storage in a computer readable storage medium withinthe respective computing/processing device.

Computer readable program instructions for carrying out operations ofthe present invention may be assembler instructions,instruction-set-architecture (ISA) instructions, machine instructions,machine dependent instructions, microcode, firmware instructions,state-setting data, or either source code or object code written in anycombination of one or more programming languages, including an objectoriented programming language such as Smalltalk, C++ or the like, andconventional procedural programming languages, such as the “C”programming language or similar programming languages. The computerreadable program instructions may execute entirely on the user'scomputer, partly on the user's computer, as a stand-alone softwarepackage, partly on the user's computer and partly on a remote computeror entirely on the remote computer or server. In the latter scenario,the remote computer may be connected to the user's computer through anytype of network, including a local area network (LAN) or a wide areanetwork (WAN), or the connection may be made to an external computer(for example, through the Internet using an Internet Service Provider).In some embodiments, electronic circuitry including, for example,programmable logic circuitry, field-programmable gate arrays (FPGA), orprogrammable logic arrays (PLA) may execute the computer readableprogram instructions by utilizing state information of the computerreadable program instructions to personalize the electronic circuitry,in order to perform aspects of the present invention.

Aspects of the present invention are described herein with reference toflowchart illustrations and/or block diagrams of methods, apparatus(systems), and computer program products according to embodiments of theinvention. It will be understood that each block of the flowchartillustrations and/or block diagrams, and combinations of blocks in theflowchart illustrations and/or block diagrams, can be implemented bycomputer readable program instructions.

These computer readable program instructions may be provided to aprocessor of a general purpose computer, special purpose computer, orother programmable data processing apparatus to produce a machine, suchthat the instructions, which execute via the processor of the computeror other programmable data processing apparatus, create means forimplementing the functions/acts specified in the flowchart and/or blockdiagram block or blocks. These computer readable program instructionsmay also be stored in a computer readable storage medium that can directa computer, a programmable data processing apparatus, and/or otherdevices to function in a particular manner, such that the computerreadable storage medium having instructions stored therein comprises anarticle of manufacture including instructions which implement aspects ofthe function/act specified in the flowchart and/or block diagram blockor blocks.

The computer readable program instructions may also be loaded onto acomputer, other programmable data processing apparatus, or other deviceto cause a series of operational steps to be performed on the computer,other programmable apparatus or other device to produce a computerimplemented process, such that the instructions which execute on thecomputer, other programmable apparatus, or other device implement thefunctions/acts specified in the flowchart and/or block diagram block orblocks.

The flowchart and block diagrams in the figures illustrate thearchitecture, functionality, and operation of possible implementationsof systems, methods, and computer program products according to variousembodiments of the present invention. In this regard, each block in theflowchart or block diagrams may represent a module, segment, or portionof instructions, which comprises one or more executable instructions forimplementing the specified logical function(s). In some alternativeimplementations, the functions noted in the block may occur out of theorder noted in the figures. For example, two blocks shown in successionmay, in fact, be executed substantially concurrently, or the blocks maysometimes be executed in the reverse order, depending upon thefunctionality involved. It will also be noted that each block of theblock diagrams and/or flowchart illustration, and combinations of blocksin the block diagrams and/or flowchart illustration, can be implementedby special purpose hardware-based systems that perform the specifiedfunctions or acts or carry out combinations of special purpose hardwareand computer instructions.

The descriptions of the various embodiments of the present disclosurehave been presented for purposes of illustration, but are not intendedto be exhaustive or limited to the embodiments disclosed. Manymodifications and variations will be apparent to those of ordinary skillin the art without departing from the scope and spirit of the describedembodiments. The terminology used herein was chosen to explain theprinciples of the embodiments, the practical application or technicalimprovement over technologies found in the marketplace, or to enableothers of ordinary skill in the art to understand the embodimentsdisclosed herein.

What is claimed is:
 1. A differential refractometer comprising: asolvent delay volume configured to be coupled to an outlet of achromatography pump; an eluent flow meter configured to be coupled to anoutlet of a chromatography column and coupled to an eluent inlet of asample cell, wherein the eluent flow meter is configured to measure aneluent flow rate of an eluent flowing through the sample cell; a solventflow regulator coupled to an outlet of the solvent delay volume andcoupled to a solvent inlet of a reference cell, wherein the solvent flowregulator is configured to measure and to regulate a solvent flow rateof a solvent flowing through the reference cell; an instrumentcontroller configured to receive the eluent flow rate from the eluentflow meter, configured to receive the solvent flow rate from the solventflow regulator, configured to receive a flow rate ratio from a flow rateratio data source, wherein the flow rate ratio indicates a ratio of theeluent flow rate to the solvent flow rate, and configured to transmit aflow command to the solvent flow regulator to achieve the flow rateratio; and an optical bench configured to measure, in response toreceiving from the instrument controller a signal indicating that theflow rate ratio has been achieved, a difference between a refractiveindex of the eluent present in the sample cell and a refractive index ofthe solvent present in the reference cell.
 2. The differentialrefractometer of claim 1 wherein the flow rate ratio is a ratio of avoid volume in the chromatography column to a void volume in the solventdelay column.
 3. The differential refractometer of claim 1 wherein theflow rate ratio is a ratio of a sum of a void volume in thechromatography column, a volume of tubing coupled to the chromatographycolumn, and a volume of the eluent flow meter, to a sum of a void volumein the solvent delay column, a volume of tubing coupled to the solventdelay column, and a volume of the solvent flow regulator.
 4. Thedifferential refractometer of claim 1 wherein the flow rate ratio is aratio of a total inclusion volume in the chromatography column to atotal inclusion volume in the solvent delay column.
 5. The differentialrefractometer of claim 1 wherein the flow rate ratio is a ratio of a sumof a total inclusion volume in the chromatography column, a volume oftubing coupled to the chromatography column, and a volume of the eluentflow meter, to a sum of a total inclusion volume in the solvent delaycolumn, a volume of tubing coupled to the solvent delay column, and avolume of the solvent flow regulator.